Herpesvirus gB-Induced Fusion between the Virion Envelope and Outer Nuclear Membrane during Virus Egress Is Regulated by the Viral US3 Kinase

Abstract

Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.Most enveloped viruses assemble their capsids in the cytoplasm and then become enveloped by budding from the plasma membrane or into cytoplasmic (CT) membranes. Viruses that assemble capsids in the nucleus face the fundamental problem of transporting capsids across the nuclear envelope (NE). Nonenveloped viruses, such as polyomaviruses, that are relatively small can move through nuclear pores, and larger nonenveloped viruses, such as adenoviruses (Ad), disrupt the NE (12, 55). Herpesvirus capsids are too large to move through nuclear pores (18), and, instead, herpesviruses encode machinery which promotes envelopment at the inner nuclear membrane (NM) followed by fusion with the outer NM (29, 52). There appears to be very rapid fusion at the NE because few enveloped herpesvirus particles are normally observed within the perinuclear space. Once present in the cytoplasm, nonenveloped capsids acquire a secondary envelope, producing virions that bud into the Golgi apparatus or trans-Golgi network (13, 29, 52). These mature virions are then secreted from cells.Some aspects of the envelopment of herpesviruses at the inner NM are known. Here, the viruses encounter a major obstacle, the nuclear lamina, a rigid network of lamin proteins lining the inner NM. Herpesviruses disrupt the nuclear lamina in order to assemble along the inner surface of the NM...

Herpes Simplex Virus 1 Glycoprotein B and US3 Collaborate To Inhibit CD1d Antigen Presentation and NKT Cell Function

“…Due to its fusogenic function, its cell surface expression must be tightly regulated (42). Recently, the viral serine/threonine kinase US3 has been found to play a key role in modulating gB cell surface expression (31,40,72), suggesting that it might collaborate with gB in affecting CD1d expression. HeLa.CD1d cells were transiently transfected with gB or US3 cDNA constructs or both, together with a peGFP-N1 plasmid to allow detection of transfected cells.…”

“…US3 is a viral serine/threonine protein kinase (31, 53, 72). To examine whether the protein kinase activity is necessary for inhibiting CD1d expression, we generated a kinase-inactive US3 mutant by mutating conserved lysine 220 to alanine as reported previously (72) and cotransfected the mutant plasmid with the gB plasmid into HeLa.CD1d cells. CD1d downregulation was no longer observed (Fig.…”

“…The HSV-1 strain 17 US3 gene was subcloned by PCR into pLPCX at a BglII/ NotI site from cosmid 48 (kindly provided by Andrew Davidson, MRC Virology, Glasgow, United Kingdom) (12). US3 K220A (US3KA) mutant was generated by PCR mutating lysine 220 (codon AAG) to alanine (codon GCG) as reported by Wisner et al (72). The US3 gene from the HSV-1 KOS strain was cloned by PCR into pcDNA3.1 vector as reported previously (45).…”

“…Some cell surface receptors, such as Memapsin 2, are phosphorylated at their di-leucine motif, and this phosphorylated motif acquires the ability to interact with Golgi-localized ␥-ear-containing ARFbinding (GGA) proteins, redirecting them from the recycling pathway to the TGN (25). gB can be directly phosphorylated by US3 protein kinase and, in fact, multiple phosphorylation sites have been discovered near the dileucine motif (40,72). It will be interesting to determine whether US3 phosphorylation of gB at these sites leads to TGN retrieval.…”

Role of tegument proteins in herpesvirus assembly and egress

“…Consistent with this notion, EBV, KSHV and PrV gB null mutants all exhibit defects in nuclear egress (Peeters et al, 1992;Lee and Longnecker, 1997;Krishnan et al, 2005). In the primary enveloped virions, pUS3 can phosphorylate the gB cytoplasmic domain and this modification is important for gB-mediated fusion of the virion envelope and the outer NM (Wisner et al, 2009). Thus, pUS3 plays important roles in both primary envelopment and deenvelopment.…”